Protein misfolding and aggregation are associated with aging, as well as a variety of human diseases, including Parkinson's disease, cystic fibrosis, amyotrophic lateral sclerosis (ALS), short chain acyl-CoA dehydrogenase (SCAD) deficiency and hypertrophic cardiomyopathy. Mitochondrial proteins are at increased risk of protein misfolding and aggregation as they are located in proximity to the respiratory chain, which is a powerful source of reactive oxygen species (ROS). When exposed to elevated ROS, mitochondrial proteins are highly susceptible to oxidative damage and conformational defects. The surveillance system that oversees mitochondrial protein quality control is composed of ATP-dependent proteases, which degrade abnormal and damaged proteins, as well as molecular chaperones, which mediate protein folding and facilitate protein degradation. Within the mitochondrial matrix the ATP-dependent proteases Lon and ClpXP selectively degrade both normal and abnormal proteins in response to metabolic demands and changing environmental conditions. The mitochondrial matrix chaperones Mortalin and Tid1, function together to fold nascent polypeptides and assist Lon and ClpXP in the degradation of misfolded proteins. In addition, our preliminary results show for the first time, that Mortalin and Tid1 also mediate protein disaggregation. The aims of this project are: (1) To elucidate the functions of Mortalin and Tid1 in protein disaggregation, reactivation and refolding, as well as the interplay between these chaperones and the mitochondrial ATP-dependent proteases Lon and ClpXP;and (2) to identify endogenous mitochondrial substrates of these proteases and chaperones. We hypothesize that these quality control proteases and chaperones function to- (a) prevent the toxic accumulation of abnormal proteins, (b) facilitate the degradation of non-native polypeptides and (c) reactivate the functional state of proteins once they are disaggregated. Our research strategy is to optimize biochemical and cell culture assay systems to study chaperone-assisted protein disaggregation, reactivation/refolding and degradation of reporter substrates. Quantitative biochemical assays will be employed to measure the ability of purified Mortalin and Tid1 to disaggregate and reactivate insoluble and enzymatically inactive glucose-6- phosphate dehydrogenase (aggG6PDH). In parallel, cell culture experiments using knockdown or overexpressing cell lines for Tid1, Lon or ClpP, will be used to determine the interplay between these quality control proteins in the unfolding and degradation of an aggregation-prone mutant of ornithine transcarbamylase (aggOTC). Lastly, a proteomics approach will be undertaken to identify endogenous mitochondrial substrates of Tid1, Lon and ClpP. This approach is supported by electron microscopy data showing the accumulation of inclusion bodies within mitochondria of Lon-depleted cells. We predict a similar phenotype in ClpP- or Tid1- depleted mitochondria. Mitochondrial proteins that accumulate or aggregate upon depletion of Lon, ClpP or Tid1 are likely to be substrates. The results obtained from this project will provide the knowledge and experimental assays needed to exploit the function of these mitochondrial chaperones and proteases in the management or treatment of protein-misfolding and possibly other diseases. PUBLIC HEALTH RELEVANCE: The aims of this R21 project are to elucidate the functions of the mitochondrial chaperones mortalin and Tid1 in protein disaggregation, refolding and reactivation, as well as their cooperative function with the mitochondrial ATP-dependent proteases Lon and ClpXP. In addition, we aim to identify endogenous mitochondrial substrates of these proteases and chaperones. The results obtained from this project will directly contribute to expanding current knowledge and experimental tools required to elucidate and exploit the function of these quality control proteins in protein misfolding and aggregation diseases such as- Parkinson's and Alzheimer's disease, cystic fibrosis, amyotrophic lateral sclerosis (ALS), short chain acyl-CoA dehydrogenase (SCAD) deficiency and hypertrophic cardiomyopathy.